W izard ^ ^ maxiprep pZLlCYP71B7 plasmid DNA from the original DHIOB host strain (Section 2.1) was prepared as described (Section 2.7), the CYP71B7 cDNA insert excised
by EcoRL/Notl restriction digestion (Section 2.8) and gel purified using 'Geneclean'
(Section 2.10). The insert was then ligated (Section 2.8) into EcoKL/Notl digested yeast expression shuttle vectors pYES2 and pGY (Section 2.2). The resulting plasmids, denoted pYES2CYP71B7 and pGYCYP71B7, were verified by gel electrophoresis (Section 2.9) which showed the ligations had been successful.
Following verification, plasmids pYES2CYP71B7 and pGYCYP71B7 were initially transformed (Section 2.8) into E.coli strain DH5a (Section 2.1) to allow amplification of the plasmids for ease of subsequent manipulation. Transformants were verified by
EcoRl/Notl digestion of W izard ^^ miniprep plasmid DNA. Plasmid DNA from colonies
which had been successfully transformed with pYES2CYP71B7 and pGYCYP71B7 was then used to transform (Section 2.8) two S. cerevisiae strains, R3 and ATTC44773 (Section 2.1). Transformants were selected on SD medium minus uracil. Negative controls for CYP71B7 P450 yeast expression were included by transformation of both S.
cerevisiae strains with pGY and pYES2 vectors without CYP71B7 inserts. Restriction
analysis of plasmid DNA prepared as described in Section 2.7 showed that pGYCYP71B7 and pYES2CYP71B7 had been successfully introduced into the S. cerevisiae strains R3 and ATTC44773, together with their respective negative controls, pYES2 and pGY.
In order to whether the transformed yeast expressed the CYP71B7 cyt P450, CO difference spectroscopy (Section 2.14) was carried out on whole yeast cells grown in liquid culture as described in Section 2.3. Whole yeast cells prepared from
ATTC44773 cells transformed with pGYCYP71B7 exhibited a faint CO difference spectrum characteristic of cyt P450 (data not shown). No such cyt P450 CO spectrum was seen with the corresponding negative control. Spectroscopic detection of cyt P450 in intact cells was difficult, with the amount of cyt P450 detected typically low. All subsequent spectroscopic analyses were therefore performed on yeast microsomal membrane fractions prepared using the methods described in Section 2.14.
Analysis of microsomal membranes allowed more definitive cyt P450 CO difference spectra to be obtained. For initial analyses, the glass bead method (Section 2.14) was used to prepare microsomal membranes. Figures 5.1 and 5.2 show the CO difference spectra exhibited by microsomes prepared from R3 and ATTC44773 cells transformed with pGYCYP71B7, and also with the pGY control. A characteristic cyt P450 CO spectrum, with an absorbance peak at 450 nm, was seen with pGYCYP71B7 in both S. cerevisiae strains, which was considerably lower or absent in the vector controls. CO difference spectroscopy on microsomes prepared from S. cerevisiae transformed with pYES2CYP71B7 showed weak P450 spectra (data not shown), but results were inconclusive and inconsistent with this vector. Galactose induction was more time consuming than growth of S. cerevisiae with the constitutive vector and the R3 S.
cerevisiae strain was found to grow better than the ATTC44773 cells. Thus, only the
pGYCYP71B7 and pGY R3 S. cerevisiae transformants were selected for use in fermentation scale-up procedures (Section 2.14) for microsomal analyses. These are referred to as 'expressing' (microsomes prepared from pGYCYP71B7 transformed S.
cerevisiae) and 'control' (microsomes prepared from pGY transformed S. cerevisiae)
microsomes in subsequent text.
membrane and therefore cyt P450 recovery. It was found that microsomes prepared using the lytic enzyme method (Section 2.14) generally gave improved cyt P450 CO spectra and higher levels o f cyt P450 than those prepared with the glass bead method (the method used to prepare the microsomes used in Figures 5.1 and 5.2). The lytic enzyme method was therefore adopted for all subsequent microsome preparations. The reason for this difference may have been due to the fact that with the glass bead method, cells are grown to a high cell density prior to harvesting. It may be that the peak for maximal activity for CYP71B7 lies at an earlier growth stage, such as that used in the lytic enzyme method. This was the case for mouse P450IA1 expressed in yeast also grown in minimal medium (Urban, Guilin, et. cd. 1990). The optimal yeast cell density for efficient P450IA1
A
expression, measured in terms of P450IA1 EROD activity, was f o u n ^ ie in a narrow
7 1
growth stage around 2 - 3 x 10 cells m l' . It would be useful therefore to investigate CYP71B7 expression levels with respect to growth stage of the host cells for future optimisation o f expression systems with the CYP71B7 cyt P450.
Analysis of CO difference spectra showed there was considerable variation in the amount of cyt P450 present in microsomes prepared from different batches of transformed R3 cells. On average, cells contained 17.46 +/- 4.6 pmol cyt P450 mg'^ microsomal protein (+/- standard error of the mean (SEM), n = 6), with a maximum of 60 pmol cyt P450 mg'^ microsomal protein in one preparation. Cyt P450 could also be detected in control microsomes prepared from R3 cells transformed with pGY, but again the level varied between batches. Cyt P450 levels were consistently lower in control microsomes than in expressing microsomes, averaging 8.0 +/- 3.3 pmol cyt P450 mg"^ microsomal protein (SEM, n = 5), with a maximum of 32 pmol mg'^ microsomal protein in one preparation. In certain batches of control microsomes, cyt P450 was barely detectable or completely absent.
A
0.002 X ) 4 0 0 4 2 0 4 4 0 4 6 0B
W a v e le n g th ( n m ) 0.002 4 2 0 4 4 0 4 0 0 4 6 0 W a v e le n g th ( n m )Figure 5,1 Cyt P450 CO difference spectrum o f microsome from S. cerevisiae (R3) transformed with pGYCYP71B7 and pG Y
Microsomes (ca 2 mg microsomal protein) prepared from p G Y C Y P llB l (A) and p G Y (B) transformed R3 cells were suspended in 20 % (v/v) glycerol, 1 m M EDTA, 10 m M Tris.Cl (pH 7.5) to 1 m l and a fe w grains o f sodium dithionite added. The suspension was split between two 0.5 ml matched quartz cuvettes and a baseline recorded between 400 and 500 nm. CO was bubbled through the front sample cuvette fo r 60 s and the spectrum between 400 and 500 nm recorded. Membranes were isolated using the glass bead method.
0 .0 0 4 u ü c ed X3 O X ) < 4 4 0 4 6 0 4 2 0 4 0 0
B
W a v e le n g th ( n m ) 0 .0 0 4 <u Ü c cd O X ) < 4 6 0 4 4 0 4 2 0 4 0 0 W a v e le n g th ( n m )F igure 5 .2 Cyt P450 CO difference spectrum o f microsomes from S.cerevisiae
(ATTC44773) transformed with pGYCYP71B7 andpG Y
Microsomes (ca 2 mg microsomal protein) prepared from pG YC YP VlB ? (A) andpG Y (B) transformedATTC44773 cells were suspended in 20 % (v/v) glycerol, 1 m M EDTA, 10 m M Tris.Cl (pH 7.5) to 1 ml and a few grains o f sodium dithionite added. The suspension was split between two 0.5 ml matched quartz cuvettes and a baseline recorded between 400 and 500 nm.CO was bubbled through the front sample cuvette fo r 60 s and the spectrum between 400 and 500 nm recorded. Membranes were isolated using the glass bead method.
An analysis o f variance, for a randomised block design with each preparation being considered as a block, showed that these means for control and expressing microsomes were significantly different (P<0.05). Figure 5.3 shows the CO difference spectra obtained for microsomes (prepared using the lytic enzyme method) from R3 cells transformed with pGYCYP71B7 and pGY. In CYP71B7 expressing microsomes the maximum absorbance occurred at 448 nm, whereas the endogenous cyt P450 isoform exhibited maximum absorbance at 452 nm. Examination of the spectrum obtained for expressing microsomes (Figure 5.3 A) shows that the absorbance peak centred at 448 nm is broad by comparison with that of control microsomes (Figure 5.3 B), with a discernable shoulder at higher wavelength. This feature implies that CYP71B7 and the endogenous yeast cyt P450 are most likely co-expressed. In previous studies with the R3 yeast strain, the spectral maximum of cyt P450 was 448 nm (Fang, Venkateswarlu, et. al. 1994), thus it was surprising therefore to observe a maximum of 452 nm in microsomes prepared from yeast transformed only with the pGY vector. However, this was observed consistently in all control microsome preparations. Overall the CO difference spectra clearly demonstrated the enhanced levels of cyt P450 seen with cells transformed with CYP71B7 in comparison to the control microsomes, (the spectra of which represent endogenous S. cerevisiae cyt P450(s)) confirming that a novel cyt P450 isoform was expressed in yeast transformed with pGYCYP71B7 at a level at least equivalent to that of endogenous cyt P450 isoforms.
448 nm
0.02
452 nm